Excess telomere shortening has been observed in most cancer cells. The telomere quantitative polymerase chain reaction (qPCR) assay has become an important tool for epidemiological studies examining the effects of aging, stress, and other factors on the length of telomeres. Current telomere qPCR methods analyze the relative length of telomeres by amplifying telomere sequence products and normalizing with single-copy gene products. However, the current telomere qPCR does not always reflect absolute telomere length in cancer DNA. Because of genomic instability in cancer cells, we hypothesized that the use of single-copy genes (scg) is less accurate for normalizing data in cancer DNA and that new primer sets are required to better represent relative telomere length in cancer DNA. We first confirmed that cancer cells had a different copy ratio among different scg, implying that DNA is aneuploid. By using the new primer sets that amplify multiple-copy sequences (mcs) throughout the genome, the telomere qPCR results showed that the mcs primers were interchangeable with the scg primers as reference primers in normal DNA. By comparing results from the traditional southern blotting method (as kilobases) and results from monochrome multiplex qPCR using the mcs primers (as T/M ratios), we verified that the T/M ratio is highly correlated with absolute telomere length from the southern blot analysis. Together, the mcs primers were able to represent the telomere lengths accurately in cancer DNA samples. These results would allow for analyses of telomeres within cancerous DNA and the development of new, less invasive diagnostic tools for cancer.